DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. DNA topology is a tightly-regulated property of the DNA double helix that affects genomic stability and influences susceptibility to cancer and certain hereditary diseases, such as fragile X syndrome and autism. DNA topoisomerases that control DNA topology inside cells are important targets of anti-cancer drugs, such as camptothecin and doxorubicin, and antibacterial agents, such as ciprofloxacin.
Typically agarose gel electrophoresis is employed to study DNA topology. Because gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. For example, fluorescence dyes, such as PicoGreen (1), have been shown to differentially bind to supercoiled (sc) and relaxed (rx) DNA molecules to yield different fluorescence properties. These fluorescence dyes were used to study DNA topoisomerases; however, the difference of the fluorescence intensity of the dyes binding to sc and rx DNA is too small to be widely used to study the properties of DNA topoisomerases and to screen inhibitors against these topoisomerases (1).
Another type of assay was developed based on a unique property of sc DNA molecules that prefer binding to triplex-form oligomers if the sc plasmids contain one or multiple triplex-forming sequences (2, 3). Maxwell and coworkers utilized a method in which an immobilized triplex-forming oligomer more efficiently captured sc plasmids than rx plasmids (2). The captured plasmids could subsequently be quantified by a DNA-binding dye, such as SYBR Green. However, this method requires immobilization of oligomer to a solid surface, filtration, and multiple washing steps. Because streptavidin-coated 1526-well plates are not commercially available, this method is not compatible with ultra-high throughput screening to identify gyrase inhibitors from small compound libraries using 1526-well plates.
Another method, also based on the triplex-forming oligomers, was developed by using fluorescence anisotropy for the readout (3). Nevertheless, the signal to noise ratio is a concern and an expensive fluorimeter with the capacity to measure fluorescence anisotropy is required (3).
More recently, Berger and coworkers made a circular plasmid DNA template that contains a fluorophore (fluorescein) and quencher (dabcyl) on opposite strands of a double-stranded DNA molecule and developed a real-time assay to study DNA topological changes with this fluorescently labeled DNA (4). However, the production involving two steps of fluorophore and quencher insertion into the DNA result in a low yield of fluorescently labeled DNA, which low yield is cost prohibitive and impedes wide use of the assay to study DNA topology, topoisomerases, and to screen compounds against DNA topoisomerases (4).